The purpose of this proposal is to continue the development of our high resolution, sensitive FISH technology and apply it to the study of RNA movements in living cells in real time. 1. To extend the technology of detecting RNA transcripts in living cells to detect single molecules in living cells. 2. To describe the movements of RNA transcripts in the cytoplasm using methods capable of single molecule detection and distinguish among various models for RNA localization. 3. To use high speed microscopic 4D imaging to follow movements of RNA transcripts in the nucleus in vivo and develop algorithms to follow the movements of hundreds of molecules over a short time window to distinguish diffusional from directed events in the nucleus. This work will result in the ability to define mechanisms which regulate the spatial aspects of RNA metabolism: RNA nuclear export, cytoplasmic transport and localization, important for the normal functioning of all eucaryotic cells.